NFAT (nuclear factor of activated T cells) is a family of Ca2+regulated transcription factors whose function is controlled at the level of dephosphorylation and nuclear-cytoplasmic shuttling. NFAT is activated upon dephosphorylation by the Ca2 calmodulin-dependent protein phosphatase calcineurin, and returned to an inactive state upon concerted rephosphorylation by 3 different kinases, CK1, DYRK and GSK3. Once in the nucleus, NFAT can pair with different transcriptional partners to activate diverse transcriptional programs in many different cell types. To test the role of NFAT in lymphocyte immune responses, we have generated hyperactivable NFAT by altering the affinity of two docking interactions made by the wildtype protein: increasing the affinity of the NFAT- calcineurin interaction, and decreasing the affinity of the NFAT-CK1 interaction. Both manipulations increase the proportion of active NFAT relative to the inactive form. NFAT is known to participate in at least five different transcriptional programmes in T cells. Cooperation of NFAT with AP-1 (Fos-Jun) is necessary for effector T cell responses; NFAT in the absence of AP-1 induces T cell anergy; cooperation of NFAT with FOXP3 promotes the suppressive function of regulatory T cells (Tregs), and cooperation of NFAT with GATA3 or Tbet drives the effector functions of Th1 and Th2 cells respectively. In this application, we take advantage of our structural information on the NFAT:AP-1:DNA, NFAT dimer:DNA and NFAT:FOXP3:DNA complexes. We have identified a mutation (R468A/I469A/T535G, abbreviated RIT) which abrogates interaction of NFAT1 with AP-1 proteins and is a powerful inducer of T cell anergy. This mutation does not affect NFAT dimer formation or the NFAT:FOXP3 interaction required for Treg function. Here we will investigate the role of NFAT proteins in Treg function and cancer. In Aim 1 we will analyse lymphocyte development and function in transgenic mice conditionally expressing hyperactivable forms of NFAT from the ROSA26 locus, and in mice with the RIT mutation knocked- in to the endogenous NFAT1 locus. We are particularly interested in whether NFAT and NFAT:AP-1 cooperation contribute to development of Foxp3-expressing regulatory T cells (Tregs). In Aim 2, we will examine the influence of NFAT proteins on tumour development. Hyperactivable or mutant NFATs will be tested for their role in tumour development and metastasis, using mouse models in which there is evidence that the calcineurin/ NFAT pathway has an important role. In Aim 3, we will determine whether T cell tolerance to tumours can be influenced by ablating the cooperation between NFAT and AP-1. We will compare the roles of NFATs lacking or bearing the RIT mutation, which greatly attenuates interaction with AP-1. PUBLIC HEALTH RELEVANCE: In this proposal we plan to investigate the role of a protein known as NFAT in cancer development and progression. NFAT is a transcription factor that binds DNA and turns on genes in many different cell types. It can directly control the growth of cancer cells and it also controls the immune responses that are designed to attack and eliminate cancers. We will express more or less powerful forms of NFAT in mice, and ask how this alters the development and progression of specific types of cancers.